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Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a powerful and sensitive method used in gene expression analysis. Suitable reference genes, which are stable under all experimental circumstances and tissues significantly improve the accuracy of qRT-PCR data. In this study, the stability of six genes, namely, 18S ribosomal RNA (18 s ), beta-actin ( actb ), elongation factor 1-alpha ( ef1α ), glyceraldehyde-3-phosphate-dehydrogenase ( gapdh ), cathepsin D ( ctsd ), and beta-2-microglobulin ( b2m ) were evaluated as potential references for qRT-PCR analysis. The genes were examined in the hypothalamus-pituitary-ovary-liver (HPOL) axis throughout turbot ovarian development via using the geNorm, NormFinder and BestKeeper algorithms. Results showed that the most stable reference genes were  ef1α ,  actb , and  ctsd  in the hypothalamus, pituitary, ovary and liver, respectively. The best-suited gene combinations for normalization were 18 s ,  ef1α , and  ctsd  in the hypothalamus;  actb ,  ctsd , and 18 s  in the pituitary;  actb , and  ctsd  in the ovary;  gapdh  and  ctsd  in the liver. Moreover, the expression profile of estrogen receptor α ( erα ) manifested no significant difference normalization to the aforementioned best-suited gene during turbot ovarian development. However, no single gene or pair of genes is suitable as an internal control and account for the amplification differences among the four tissues during ovarian development. In summary, these results provide a basic data for the optimal reference gene selection and obtain highly accurate normalization of qRT-PCR data in HPOL axis-related gene expression analysis during turbot ovarian development.

Reference gene validation for quantification of gene expression during ovarian development of turbot (Scophthalmus maximus)